This invention relates to cancer detection and treatment and, more particularly, to a novel protein called xe2x80x9cp19ARF protein,xe2x80x9d xe2x80x9cp19ARFxe2x80x9d, xe2x80x9cARF-p19xe2x80x9d or simply xe2x80x9cARFxe2x80x9d that is involved in regulation of the eukaryotic cell cycle. Protein ARF-p19 is encoded by a nucleic acid derived from the gene, INK4A, which also encodes an inhibitor of D-type cyclin-dependent kinases called xe2x80x9cp16InK4a protein,xe2x80x9d xe2x80x9cp16InK4axe2x80x9d or simply xe2x80x9cInK4a-p16.xe2x80x9d
Transcripts encoding InK4a-p16 originate from a first promoter, E1a; the present invention is based on the observation that some INK4A transcripts initiate from a second promoter, E1b, and contain an Alternative Reading Frame, ARF, which overlaps the InK4a-p16 reading frame to some degree. ARF transcripts direct the production of a protein that has ARF-p19 amino acid sequences instead of the previously-known InK4a-p16 sequences. Like InK4a-p16, ARF-p19 regulates the eukaryotic cell cycle. When overexpressed, ARF-p19 inhibits cells from proceeding past both the G1 and G2 phases of the cell cycle. However, the mechanism(s) by which ARF-p19 acts are unlike those of InK4a-p16, which acts by directly and specifically interacting with CDK (cyclin D-dependent kinase) proteins and thus preventing CDK-cyclin D interactions.
In addition to (1) ARF-p19 proteins, this invention further relates to (2) nucleic acids that encode ARF-p19 isolated from mice, humans and other mammals; (3) antibodies that specifically bind ARF-p19 protein or polypeptides derived therefrom; (4) methods for detecting one or more nucleic acids encoding ARF-p19, or alterations in such nucleic acids; (5) methods for producing ARF-p19 proteins using nucleic acids that encode ARF-p19; (6) purified ARF-p19 proteins, or fusion proteins derived from the joining of an ARF-p19 polypeptide sequence with a second polypeptide sequence; (7) methods of treating cancer using purified ARF-p19 proteins or fusion proteins derived therefrom; (8) methods of inducing cell cycle arrest using ARF-p19 proteins or nucleic acids encoding ARF-p19 proteins; (9) methods for detecting ARF-p19 proteins using antibodies that specifically bind ARF-p19 proteins; (10) methods of selectively killing cells having uncontrolled growth using antibodies that specifically bind ARF-p19 proteins, or conjugates derived from such antibodies; (11) methods of stimulating cell growth using antibodies that specifically bind ARF-p19 proteins, or fragments derived from such antibodies; and (12) transgenic non-human animals that have a genetically engineered alteration in one or more nucleic acids encoding ARF-p19 proteins but which express normal levels of wild-type InK4a-p16 protein, or which overexpress human ARF-p19 or mutant forms of ARF-p19.
Neoplasia, the pathological process by which tumors develop, necessarily involves unregulated, or at best misregulated, cellular growth and division. The molecular pathways that regulate cellular growth must inevitably intersect with those that regulate the cell cycle. The cell cycle consists of a cell division phase and the events that occur during the period between successive cell divisions, known as interphase. Interphase is composed of successive G1, S, and G2 phases, and normally comprises 90% or more of the total cell cycle time. Most cell components are made continuously throughout interphase; it is therefore difficult to define distinct stages in the progression of the growing cell through interphase. One exception is DNA synthesis, since the DNA in the cell nucleus is replicated only during a limited portion of interphase. This period is denoted as the S phase (S=synthesis) of the cell cycle. The other distinct stage of the cell cycle is the cell division phase, which includes both nuclear division (mitosis) and the cytoplasmic division (cytokinesis) that follows. The entire cell division phase is denoted as the M phase (M=mitotic). This leaves the period between the M phase and the start of DNA synthesis, which is called the G1 phase (G=gap), and the period between the completion of DNA synthesis and the next M phase, which is called the G2 phase (Alberts, B. et al., Molecular Biology of the Cell, Garland Publishing, Inc., New York and London (1983), pages 611-612.).
Progression through different transitions in the eukaryotic cell cycle is positively regulated by a family of master enzymes, the cyclin-dependent kinases (reviewed by Sherr, C. J., Cell 73:1059-1065 (1993)). These holoenzymes are composed of two proteins, a regulatory subunit (the cyclin), and an associated catalytic subunit (the actual cyclin-dependent kinase or CDK), the levels of which vary with different phases of the cell cycle (Peters, G., Nature 371:204-205 (1994)). Both cyclins and CDKs represent molecular families that encompass a variety of genetically related but functionally distinct proteins. Generally, different types of cyclins are designated by letters (i.e., cyclin A, cyclin B, cyclin D, cyclin E, etc.); CDKs are distinguished by numbers (CDK1, CDK2, CDK3, CDK4, CDK5, etc.; CDK1 is a.k.a. CDC2).
CDK-cyclin D complexes regulate the decision of cells to replicate their chromosomal DNA (Sherr, Cell 73:1059-1065 (1993)). As cells enter the cycle from quiescence, the accumulation of CDK-cyclin D holoenzymes occurs in response to mitogenic stimulation, with their kinase activities being first detected in mid-G1 phase and increasing as cells approach the G1/S boundary (Matsushime et al., Mol. Cell. Biol. 14:2066-2076 (1994); Meyerson and Harlow, Mol. Cell. Biol. 14:2077-2086 (1994)). The cyclin D regulatory subunits are highly labile, and premature withdrawal of growth factors in G1 phase results in a rapid decay of CDK-cyclin D activity that correlates with the failure to enter S phase. In contrast, removal of growth factors late in GI phase, although resulting in a similar collapse of CDK-cyclin D activity, has no effect on further progression through the cell cycle (Matsushime et al., Cell 65:701-713 (1991)). Microinjection of antibodies to cyclin D1 into fibroblasts during G1 prevents entry into the S phase, but injections performed at or after the G1xe2x86x92S transition are without effect (Baldin et al., Genes and Devel. 7:812-821 (1993); Quelle et al., Genes and Devel. 7:1559-1571 (1993)). Therefore, CDK-cyclin D complexes execute their critical functions at a late G1 checkpoint, after which cells become independent of mitogens for completion of the cycle. In mammals, cells enter the cell cycle and progress through G1 phase in response to extracellular growth signals which trigger the transcriptional induction of D-type cyclins. The accumulation of D cyclins leads to their association with two distinct catalytic partners, CDK4 and CDK6, to form kinase holoenzymes. Several observations argue for a significant role of the cyclin D-dependent kinases in phosphorylating the retinoblastoma protein, pRb, leading to the release of pRB-associated transcription factors that are necessary to facilitate progression through the G1xe2x86x92S transition. First, CDK-cyclin D complexes have a distinct substrate preference for pRb but do not phosphorylate the canonical CDK substrate, histone H1 (Matsushime et al., Cell 71:323-334 (1992); Matsushime et al., Mol. Cell. Biol. 14:2066-2076 (1994); Meyerson and Harlow, Mol. Cell. Biol. 14:2077-2086 (1994)). Their substrate specificity may be mediated in part by the ability of D-type cyclins to bind to pRb directly, an interaction which is facilitated by a Leu-X-Cys-X-Glu pentapeptide that the D cyclins share with DNA oncoproteins that also bind pRb (Dowdy et al., Cell 73:499-511 (1993); Ewen et al., Cell 73:487-497 (1993); Kato et al., Genes and Devel. 7:331-342 (1993)). Second, cells in which pRb function has been disrupted by mutation, deletion, or after transformation by DNA tumor viruses are no longer inhibited from entering S phase by microinjection of antibodies to D cyclin, indicating that they have lost their dependency on the cyclin D-regulated G1 checkpoint (Lukas et al., J. Cell. Biol. 125:625-638 (1994); Tam et al., Oncogene 9:2663-2674 (1994)). However, introduction of pRb into such cells restores their requirement for cyclin D function (Lukas et al., J. Cell. Biol. 125:625-638 (1994)). Third, pRb-negative cells synthesize elevated levels of a 16 kDa polypeptide inhibitor of CDK4, xe2x80x9cp16InK4axe2x80x9d (a.k.a. xe2x80x9cInK4a-p16xe2x80x9d or simply xe2x80x9cp16xe2x80x9d), which is a member of a recently discovered class of cell cycle regulatory proteins (Nasmyth and Hunt, Nature 366:634-635 (1993); Peters, G., Nature 371:204-205 (1994)) and which is found in complexes with CDK4 at the expense of D-type cyclins during G1 phase (Bates et al., Oncogene 9:1633-1640 (1994); Serrano et al., Nature 366:704-707 (1993); Xiong et al., Genes and Devel. 7:1572-1583 (1993)). The fact that such cells cycle in the face of apparent CDK4 inhibition again implies that D-type cyclins are dispensable in the Rb-negative setting.
The InK4 gene family (xe2x80x9cInK4xe2x80x9d signifies Inhibitors of CDK4) is known to include at least three other low molecular weight polypeptides, InK4b-p15, induced in human epithelial cells treated by transforming growth factor-xcex2 (TGF-xcex2) (Hannon, G. J., and Beach, D., Nature 371:257-261 (1994)), InK4d-p19 (Hirai, H., et al., Mol. Cell. Biol. 15:2672-2681 (1995)) and InK4c-p18 (Guan et al., Genes and Develop. 8:2939-2952 (1994); Hirai, H., et al., Mol. Cell. Biol. 15:2672-2681 (1995)). InK4d-p19 and InK4c-p18 are described in detail in U.S. Ser. No. 08/384,106, filed Feb. 6, 1995, which is hereby incorporated by reference.
Members of the InK4 family are typically composed of repeated ankyrin motifs, each of about 32 amino acids in length. All known members of the InK4 family act to specifically inhibit enzymatic activities of D-type cyclin-dependent kinases such as CDK4 and CDK6. Unlike other universal CDK inhibitors, such as p21Cip/Waf1 (El-Deiry et al., Cell 75:817-825 (1993); Gu et al., Nature 366:707-710 (1993); Harper et al., Cell 75:805-816 (1993); Xiong et al., Nature 366:701-704 (1993)) and p27KP1 (Polyak et al., Genes and Devel. 8:9-22 (1994); Polyak et al., Cell 78:59-66 (1994); Toyoshima and Hunter, Cell 78:67-74 (1994)), the InK4 proteins selectively inhibit the activities of CDK4 and CDK6, but do not inhibit the activities of other CDKs (Guan et al., Genes and Devel. 8:2939-2952 (1994); Hannon and Beach, Nature 371:257-261 (1994); Serrano et al., Nature 366:704-707 (1993)).
Like many CDK inhibitors (CKIs) (Nasmyth and Hunt, Nature 366:634-635 (1993)), InK4 family members negatively regulate progression through the mammalian cell cycle, in part in response to anti-proliferative extracellular signals. The InK4 proteins, by inhibiting the activities of a specific class of the D-type cyclin-dependent kinases (i.e., CDK4 and/or CDK6), arrest cell cycle progression in G1 phase and thus prevent cells from replicating their chromosomal DNA. Thus, in contradistinction to the positive regulation of D-type cyclin synthesis by growth factors, extracellular inhibitors of G1 progression can negatively regulate the activity of D-type cyclin-dependent kinases by inducing InK4 proteins.
Mullis et al., U.S. Pat. No. 4,965,188 (Oct. 23, 1990), describe methods for amplifying nucleic acid sequences using the polymerase chain reaction (PCR).
Beach, published PCT patent application WO 92/20796 (Nov. 26, 1992), describes genes encoding D cyclins and uses thereof.
Berns, U.S. Pat. No. 5,174,986 (Dec. 29, 1992), describes methods for determining the oncogenic potential of chemical compounds using a transgenic mouse predisposed to develop T-cell lymphomas.
Crissman et al., U.S. Pat. No. 5,185,260 (Feb. 9, 1993), describe methods for distinguishing and selectively killing transformed (neoplastic) cells using synthetic G1 kinase inhibitors.
Stone, S., et al., Cancer Research 55:2988-2994 (1995), describe two cDNAs derived from the human INK4A gene, including an xe2x80x9ca formxe2x80x9d encoding InK4a-p16 and a xe2x80x9cb formxe2x80x9d that includes an open reading frame (designated xe2x80x9cORF 2xe2x80x9d) that overlaps the reading frame encoding the ARF-p19 protein described herein. Stone et al. state that it xe2x80x9cis unknown if ORF 2 encodes a proteinxe2x80x9d (legend to FIG. 1, page 2990) and indicate that xe2x80x9cORF 2 has not been selectively maintained and probably does not encode a proteinxe2x80x9d (page 2989, column 2, lines 20-21).
Mao, L., et al., Cancer Research 55:2995-2997 (1995), describe two transcripts and corresponding cDNAs derived from the human INK4A gene, designated xe2x80x9cp16xe2x80x9d and xe2x80x9cp16b.xe2x80x9d The p16 transcript is stated to encode the InK4a-p16 protein, while the p16b transcript is stated to contain a xe2x80x9ctheoretical open reading framexe2x80x9d (page 1996, column 1, line 47) that is not further defined, and suggest this sequence xe2x80x9cprobably represents an untranslated open reading framexe2x80x9d (page 2997, column 2, lines 9-10). Mao et al. state that the in vitro transcription and translation (TNT) product of the p16b cDNA is recognized by an antibody to InK4a-p16 polypeptide sequences (page 2997, column 1, lines 6), suggesting that the p16b transcript encodes an amino-terminal truncated InK4a-p16 polypeptide rather than a protein having, as ARF-p19 does, an amino acid sequence unrelated to that of InK4a-p16. However, Mao et al. also state that, using InK4a-p16 antiserum, they are unable to identify an amino-terminal truncated p16b protein in cell lines (page 2997, column 2, lines 1-2). Thus, Mao et al. are silent regarding the ARF-p19 protein described herein.
The present invention relates to the discovery in mammalian cells of a novel cell cycle regulatory protein, having a predicted molecular mass of 19 kDa, here designated xe2x80x9cARF-p19 proteinxe2x80x9d, xe2x80x9cARF-p19xe2x80x9d or simply xe2x80x9cARFxe2x80x9d. In particular, the invention relates to ARF-p19 proteins isolated from cells derived from a mouse or a human. Although derived from the gene encoding the previously-known InK4a-p16 protein, ARF-p19 arises by differential transcription and translation of InK4a-p16 sequences. That is, ARF-p19 is encoded by an alternative reading frame (ARF) and the full length protein has an amino acid sequence (SEQ ID NO:2; SEQ ID NO:4) that is wholly unrelated to that of InK4a-p16. Surprisingly, however, ARF-p19 protein functions to regulate the cell cycle in a similar but less specific manner than, and by a mechanism distinct from that of, InK4a-p16 protein.
Thus, one aspect of the invention is directed to methods of using the ARF-p19 proteins or active fragments thereof (such as the peptide encoded by exon 1xcex2) of the invention to inhibit the growth of cancer cells and/or to prevent cancer cells from replicating their chromosomal DNA. Both InK4-p16 and InK4-p15 appear to act as tumor suppressors (Noburi, T. et al., Nature 368:753-756 (1994); Kamb, A. et al., Science 264:436-440 (1994)). The genes encoding p16 and p15 map in a tandem array to the short arm of human chromosome 9 within a region that is frequently deleted in cancer cells, and the resulting loss of their anti-proliferative functions can contribute to tumorigenesis (Noburi et al., Nature 368:753-756 (1994); Okuda, T., et al., Blood 85:2321-2330 (1995)). The novel ARF-p19 protein described herein (1) plays a role in preventing the G1xe2x86x92S and G2xe2x86x92M phase transitions in normal mammalian cells, and (2) if having reduced or altered activity due to one or more mutations affecting the alternative reading frame encoding ARF-p19, could contribute to oncogenesis in some cancers, even if such mutations have no effect on the reading frame encoding InK4a-p16. Indeed, as described herein, ARF-p19 and active fragments thereof can act as tumor suppressors.
In another aspect, the invention provides nucleic acid sequences encoding ARF-p19 polypeptides and active fragments thereof from mice, humans and other mammals. The nucleic acid sequences of the invention may be expressed in the form of isolated nucleic acids, such as cDNA clones, genomic DNA clones, mRNA transcribed from either cDNA or genomic DNA clones, synthetic oligonucleotides, and/or synthetic amplification products resulting from PCR, and may be single-stranded or double-stranded.
In a related aspect, the invention provides methods for detecting nucleic acids encoding wild-type or mutant ARF-p19 proteins and active fragments thereof using the nucleic acid sequences of the invention described above. The detection of point mutations, deletions of, or other mutations in, the reading frame encoding ARF-p19 is predictive of a predisposition to, or diagnostic of, certain types of cancer.
In another related aspect, the DNA molecules of the invention described above may be cloned into expression vectors and placed in an appropriate host in order to produce ARF-p19 proteins, active fragments thereof or fusion proteins containing ARF-p19 polypeptide sequences or active fragments thereof. When placed in an animal that has cancer, this aspect of the invention relates to gene therapy for certain types of cancers.
In another aspect, the invention provides antibody compositions that bind specifically to ARF-p19 proteins and/or polypeptides derived therefrom. The antibody compositions of the invention may be polyclonal, monoclonal, or monospecific. Although all of the antibody compositions of the invention bind specifically to ARF-p19, some compositions bind to a specific epitope of ARF-p19 and thereby inhibit a specific function of ARF-p19.
In a related aspect, the invention provides methods for detecting ARF-p19 proteins using the antibody compositions described above. The detection of reduced amounts of, or altered forms of, ARF-p19 proteins is predictive of a predisposition to, or diagnostic of, certain types of cancer.
In another aspect, the invention provides transgenic non-human animals which have one or more mutations in the endogenous reading frame encoding ARF-p19, wherein said mutation results in the production of a mutant ARF-p19 protein or results in a loss of ARF-p19 expression but does not significantly affect the InK4a-p16 gene product or expression thereof. Additionally or alternatively, the transgenic non-human animals of the invention express a human wild-type or mutant ARF-p19. Because of the transgene(s) introduced into the genome of the non-human animals of the invention, the animals have a reduced and/or altered ARF-p19 activity compared to wild-type animals, and consequentially develops certain types of cancers, particularly melanomas, in a reproducible and thus predictable manner.
In a related aspect, compositions are evaluated for their potential to enhance or inhibit certain types of cancers, particularly melanomas, using the transgenic non-human animals of the invention.
In addition the present invention provides a complex, preferably an isolated complex, comprising p53, or a fragment thereof, bound to a ARF-p19 peptide or protein comprising at least 10, preferably at least 25, and more preferably at least 62 contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In a related embodiment, the complex comprises p53, or a fragment thereof, bound to a peptide or protein comprising at least 10, preferably at least 25, and more preferably at least 62 contiguous amino acid residues of the amino acid sequence of amino acids 1-84 of SEQ ID NO:2 or amino acids 1-84 of SEQ ID NO:4. In still another embodiment the complex comprises p53, or a fragment thereof, bound to a peptide or protein comprising at least 10, preferably at least 25, and more preferably 62 contiguous amino acid residues of the amino acid sequence of amino acids 1-62 of SEQ ID NO:2 or amino acids 1-62 of SEQ ID NO:4. In yet another embodiment, the complex comprises p53, or a fragment thereof, bound to the peptide encoded by exon 1xcex2 of the INKa-ARF locus of the mouse ARF-p19 (SEQ ID NO:2) or the human ARF-p19 (SEQ ID NO:4). In a particular embodiment the complex further comprises mdm2 and/or an oligonucleotide. p53 or the p53 fragment, or the ARF-p19 peptide or protein also can be a part of a fusion protein. In addition, any of the proteins and peptides included in the complexes can be recombinant proteins.
The present invention also provides methods of identifying an agent that modulates the binding of ARF-p19 and p53. One such method comprises contacting ARF-p19 or an ARF-p19 fragment with p53 or p53 fragment in the presence of a candidate agent, and determining the binding of p53 or the p53 fragment with ARF-p19 or the ARF-p19 fragment. The ARF-p19 fragment binds to p53 in the absence of the agent, and the p53 fragment binds to ARF-p19 in the absence of the agent. Preferably, the ARF-p19 fragment binds to the p53 fragment in the absence of the agent. When the binding of p53 or the p53 fragment with ARF-p19 or the ARF-p19 fragment is modulated, a candidate agent is identified as an agent that modulates the binding of ARF-p19 with p53. In one such embodiment, when the modulation of the binding of p53 or the p53 fragment with ARF-p19 or the ARF-p19 fragment leads to a decrease in the binding affinity, the agent is identified as an inhibitor of p53 binding with ARF-p19. In another such embodiment, when the modulation of the binding of p53 or the p53 fragment with ARF-p19 or the ARF-p19 fragment leads to an increase in the binding affinity, the agent is identified as an agonist of p53 binding with ARF-p19. In a particular embodiment the ARF-p19 fragment is a peptide comprising at least 10, preferably at least 25, and more preferably at least 62 contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In another embodiment the ARF-p19 fragment is encoded by exon 1xcex2 of the INKa-ARF locus of the mouse ARF-p19 (SEQ ID NO:2) or the human ARF-p19 (SEQ ID NO:4). In a related embodiment the ARF-p19 or ARF-p19 fragment is part of a fusion protein. In addition, any of the proteins and peptides can be recombinant proteins.
The present invention also provides compounds that interact with Hdm2 and Mdm2 in a manner that is analogous to the interaction of ARF-p19 with Hdm2 as disclosed below (see Example 11). Such compounds bind Dm2 (e.g., Hdm2 or Mdm2) in a mammalian cell and aid in the translocation of Dm2 to the nucleolus of the cell. Preferably the compound can induce cell-cycle arrest in a mammalian cell. These compounds include but are not limited to ARF-p19 proteins; active fragments of ARF-p19, such as a peptide comprising amino acid residues 1-37 of SEQ ID NO:2; and related compounds, i.e., derivatives thereof such as peptides that comprise specific amino acid sequences from ARF-p19 that are in peptide linkages with other ARF-p19 amino acid sequences and/or non-ARF-p19 sequences (including nucleolus translocating amino acid sequences from other proteins) thereby forming new sequences that are not found in nature. Such compounds preferably consist of 10 to 50 amino acid residues, and more preferably 5 to 40 amino acids.
In one embodiment the peptide comprises the amino acid sequence of SEQ ID NO:47 (ARG ARG PHE LEU VAL THR). In another important the peptide comprises SEQ ID NO:48 (ARG ARG PRO ARG). Preferably the peptide comprises both SEQ ID NO:47 and SEQ ID NO:48. In a related embodiment the peptide comprises amino acid residues 1-14 of SEQ ID NO:2. In another embodiment the peptide comprises amino acid residues 26-37 of SEQ ID NO:2. In still another embodiment the peptide comprises amino acid residues 82-101 of SEQ ID NO:4. In yet another embodiment the peptide comprises amino acid residues 2-14 or 1-14 of SEQ ID NO:4. In still other embodiments, the peptide comprises two or more of these sequences. Preferably, the peptide comprises one of these amino acid sequences that bind to Hmd2 and another amino acid sequence that can translocate Hmd2 to the nucleolus (as exemplified in Example 11, below). In a particular embodiment the peptide comprises the amino acid residues 1-101 of SEQ ID NO:4. In a preferred embodiment, the peptide comprises amino acid residues 1-37 of SEQ ID NO:2. It is important to note, however, that whereas the peptide comprising amino acid residues 1-37 of SEQ ID NO:2 is derived from a murine protein sequence, this peptide has been shown in Example 11 to interact with human Dmp2 (Hdmp2) and can therefore be used as a pharmaceutical/therapeutic in humans. The peptides of the presence invention can further comprise amino acid sequences from ARF-p19 comprising one or more conservative amino acid substitutions.
The present invention also includes fusion proteins or peptides that comprise the peptides of the present invention, and nucleic acids that encode the peptides and fusion peptides of the present invention. In a preferred embodiment, the nucleic acid that encodes the peptide that consists of amino acid residues 1-37 of SEQ ID NO:2 and has the nucleotide sequence of SEQ ID NO:42.
The present invention also provides the fragment of Hmd2 that binds ARF-p19 which comprises amino acid residues 210-304 of SEQ ID NO:44. The present invention further provides the nucleic acid encoding that peptide.
As stated below, the present invention further provides expression constructs for the nucleic acids of the present invention, vectors comprising the constructs and cells comprising the vectors. The present invention further provides methods of expressing the peptides from the constructs, methods of purifying the products as well as the isolated recombinant peptides themselves.
The present invention further provides methods of preventing abnormal cell growth. In one such embodiment the method comprises administering an effective amount of ARF-p19 or a ARF-p19 fragment or a related compound as described above, that can act as a tumor suppressor in a cell. In a preferred embodiment, the cell is responding to an hyperproliferative signal and the cell contains a functional (e.g., a wildtype) p53. In one embodiment of this type the hyperproliferative signal is due to an oncogene. In a particular embodiment of this type the oncogene is MYC. In one embodiment the ARF-p19 fragment is a peptide comprising at least 10, preferably at least 25, and more preferably at least 62 contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In a preferred embodiment the ARF-p19 fragment is encoded by exon 1xcex2 of the INKa-ARF locus of the mouse ARF-p19 (SEQ ID NO:2) or the human ARF-p19 (SEQ ID NO:4). In a more preferred embodiment the peptide consists of amino acid residues 1-37 of SEQ ID NO:2. In a related embodiment the ARF-p19 or ARF-p19 fragment or the related compound as described above is part of a fusion protein. In addition, any of the proteins and peptides can be recombinant proteins. The ARF-p19 or ARF-p19 fragment or a related compound as described above can be administered to the cell either by contacting the protein or peptide to the cell, or alternatively by introducing an expression vector into the cell that encodes the ARF-p19 or ARF-p19 fragment or a related compound as described above, and that expresses an effective amount of the protein or peptide.
The present invention also provides methods of treating an animal (preferably a mammal) that has a tumor and/or cancer. One such method includes administering an effective amount of a pharmaceutical composition that comprises a pharmaceutically acceptable carrier and ARF-p19 or a ARF-p19 fragment or a related compound as described above that can act as a tumor suppressor in the cell. In a preferred embodiment, the animal contains a cell that is responding to an hyperproliferative signal and the cell contains a functional (e.g., a wildtype) p53. In one embodiment of this type the hyperproliferative signal is due to an oncogene. In a particular embodiment of this type the oncogene is MYC. In one embodiment the ARF-p19 fragment is a peptide comprising at least 10, preferably at least 25, and more preferably at least 62 contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In a preferred embodiment the ARF-p19 fragment is encoded by exon 1xcex2 of the INKa-ARF locus of the mouse ARF-p19 (SEQ ID NO:2) or the human ARP-p19 (SEQ ID NO:4). In a more preferred embodiment the peptide consists of amino acid residues 1-37 of SEQ ID NO:2. In a related embodiment the ARF-p19 or ARF-p19 fragment or the related compound as described above is part of a fusion protein. In addition, any of the proteins and peptides can be recombinant proteins. The present invention also provides corresponding pharmaceutical compositions comprising an ARF-p19 or an ARF-p19 fragment or a related compound as described above that can act as a tumor suppressor; and a pharmaceutically acceptable carrier. Alternatively the ARF-p19 or ARF-p19 fragment or a related compound can be administered by introducing an expression vector into the animal that encodes the ARF-p19 or ARF-p19 fragment or the related compound and expresses an effective amount of the protein or peptide.
Also part of the present invention is an in vitro method for monitoring a therapeutic treatment of a tumor and/or cancer in an animal, preferably a mammalian subject. One such embodiment comprises evaluating the levels of ARF-p19 or an active ARF-p19 fragment in a series of biological samples obtained at different time points from a mammalian subject undergoing a therapeutic treatment for the tumor or cancer.
The present invention further provides methods of identifying an agent that can act as a tumor supressor in a cell. In one such embodiment the agent is contacted with the cell and the amount of cellular proliferation is determined. A decrease in cellular proliferation of the cell in the presence of the agent is indicative that the agent is a tumor suppressor. In a particular embodiment of this type the cell contains an homozygous disruption in its endogenous exon 1xcex2 of the INK4a-ARF locus. In one such embodiment the INK4a exon 1xcex1 and the tandemly linked INK4b locus remain intact, so that the cell does not express endogenous exon 1xcex2, but can express p16INK4a and functional (e.g., wildtype) p53). In a preferred embodiment, the animal contains a cell that is responding to an hyperproliferative signal. In one embodiment of this type the hyperproliferative signal is due to an oncogene. In a particular embodiment of this type the oncogene is MYC.
The present invention further provides a method of identifying an agent that can stimulate the apoptosis of cells. One such method comprises culturing the cells in a serum-free medium in the presence and absence of the agent and determining the amount of apoptosis of the cells. An agent is selected as stimulating apoptosis when the amount of apoptosis in the presence of the agent is greater than in its absence. In a particular embodiment of this type the cell contains an homozygous disruption in its endogenous exon 1xcex2 of the INK4a-ARF locus. In one such embodiment the INK4a exon 1xcex1 and the tandemly linked INK4b locus remain intact, so that the cell does not express endogenous exon 1xcex2, but can express p16INK4a and functional (e.g., wildtype) p53. In a preferred embodiment, the animal contains a cell that is responding to an hyperproliferative signal. In one embodiment of this type the hyperproliferative signal is due to an oncogene. In a particular embodiment of this type the oncogene is MYC. In a preferred embodiment of this type the method further comprises the step of introducing the oncogene into the cells by a viral vector. In a related embodiment the method further includes the step of inducing the expression of the oncogene by culturing the cells containing the viral vector with an inducer of the expression of the oncogene prior to culturing the cells in a serum-free medium.
As indicated above, the present invention provides transgenic knockout animals which are missing a functional ARF-p19. One such embodiment comprises a homozygous disruption in the endogenous exon 1xcex2 of the INK4a-ARF locus of the animal. The resulting knockout animal is particularly susceptible to developing spontaneous tumors. In one such embodiment the INK4a exon 1xcex1 and the tandemly linked INK4b locus remain intact, so that the cell does not express endogenous exon 1xcex2, but can express p16INK4a and functional (e.g., wildtype) p53. In a particular embodiment the transgenic knockout animal is a knockout mouse. The present invention also provides cultured cell lines derived from the knockout animals of the present invention. In addition, the present invention provides an embryo fibroblast comprising a homozygous disruption in its endogenous exon 1xcex2 of the INK4a gene. In one such embodiment the INK4a exon 1xcex1 and the tandemly linked INK4b locus remain intact, so that the cell does not express endogenous exon 1xcex2, but can express p16INK4a and functional (e.g., wildtype) p53. In a preferred embodiment of this type the embryo fibroblast is a mouse embryo fibroblast.
The present invention further provides a method for diagnosing a cell sample comprising a cell suspected of being cancerous or prone to becoming cancerous due to a mutation, deletion, or insertion in an endogenous nucleic acid encoding ARF-p19. One such embodiment comprises preparing a nucleotide sample from the cell and detecting the mutation, the deletion, or the insertion with a test nucleic acid having the nucleotide sequence of SEQ ID NO:1 or a portion thereof, or SEQ ID NO:3 or a portion thereof. When the mutation, the deletion, or the insertion is detected, the presence of the mutation, the deletion, or the insertion of the endogenous nucleic acid encoding ARP-p19 is diagnosed. In one embodiment a DNA sample is prepared. In a related embodiment an RNA sample is prepared. In a preferred embodiment the portion of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 is derived from a nucleotide sequence that is not found in p16-InK4a mRNAs. In one embodiment, the test nucleic acid is a nucleotide probe that can be used to hybridize with the nucleotide sample. In another embodiment, the test nucleic acid is a nucleic acid primer that can be used in PCR analysis. In a preferred embodiment the cell is suspected of responding to an hyperproliferative signal. In one embodiment of this type the hyperproliferative signal is suspected to be due to an oncogene. In a particular embodiment of this type the oncogene is MYC.